The goals of the proposed research are to determine the structural organization of the genes which code for enzymes of the glycolytic pathway and to elucidate the mechanism(s) involved in coordinate expression of these genes. Recombinant plasmid DNAs containing genomic DNA sequences which code for yeast glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, enolase and phosphoglycerate mutase have been isolated in this laboratory and will be fundamental to the proposed investigations. Hybrid plasmid DNAs containing structural genes which code for additional glycolytic enzymes will be isolated using complementary DNA synthesized from fractionated glycolytic messenger RNAs as hybridization "probes" for identifying E.coli K12 transformants which contain these hybrid plasmid DNAs. The precise location and size of the glycolytic structural genes within the isolated hybrid plasmid DNAs will be determined by restriction endonuclease and transcriptional mapping analysis. The possibility that genes coding for specific glycolytic enzymes are linked or share common regulatory sequences will be investigated by heteroduplex analysis and nucleotide sequence determination. It is proposed that stable glycolytic structural gene mutants be generated by transformation of competent yeast cells with isolated hybrid plasmids containing small deletions within the structural gene portion of the plasmids. Mutants which have recombined the altered gene in place of the wild type allele will be selected from the collection of transformants. The biological activity of isolated structural gene sequences will be investigated by subsequent transformation of the isolated mutants. Isolated genes coding for yeast glycolytic enzymes, as well as yeast ribosomal genes, will be transcribed in vitro with purified RNA polymerases and selective initiation and termination of RNA synthesis will be investigated. The long term goal of these studies is the eventual reconstitution of selective transcription of specific eucaryotic genes from purified components.